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A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

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A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

Mol Cell. 2021 Jan 04;:

Authors: Shah M, Taylor VL, Bona D, Tsao Y, Stanley SY, Pimentel-Elardo SM, McCallum M, Bondy-Denomy J, Howell PL, Nodwell JR, Davidson AR, Moraes TF, Maxwell KL

Abstract
The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.

PMID: 33412111 [PubMed - as supplied by publisher]



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A practical guide to cancer subclonal reconstruction from DNA sequencing.

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A practical guide to cancer subclonal reconstruction from DNA sequencing.

Nat Methods. 2021 Jan 04;:

Authors: Tarabichi M, Salcedo A, Deshwar AG, Ni Leathlobhair M, Wintersinger J, Wedge DC, Van Loo P, Morris QD, Boutros PC

Abstract
Subclonal reconstruction from bulk tumor DNA sequencing has become a pillar of cancer evolution studies, providing insight into the clonality and relative ordering of mutations and mutational processes. We provide an outline of the complex computational approaches used for subclonal reconstruction from single and multiple tumor samples. We identify the underlying assumptions and uncertainties in each step and suggest best practices for analysis and quality assessment. This guide provides a pragmatic resource for the growing user community of subclonal reconstruction methods.

PMID: 33398189 [PubMed - as supplied by publisher]



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Hyperspectral super-resolution imaging with far-red emitting fluorophores using a thin-film tunable filter.

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Hyperspectral super-resolution imaging with far-red emitting fluorophores using a thin-film tunable filter.

Rev Sci Instrum. 2020 Dec 01;91(12):123703

Authors: Vissa A, Giuliani M, Kim PK, Yip CM

Abstract
New innovations in single-molecule localization microscopy (SMLM) have revolutionized optical imaging, enabling the characterization of biological structures and interactions with unprecedented detail and resolution. However, multi-color or hyperspectral SMLM can pose particular challenges which affect image quality and data interpretation, such as unequal photophysical performance of fluorophores and non-linear image registration issues, which arise as two emission channels travel along different optical paths to reach the detector. In addition, using evanescent-wave based approaches (Total Internal Reflection Fluorescence: TIRF) where beam shape, decay depth, and power density are important, different illumination wavelengths can lead to unequal imaging depth across multiple channels on the same sample. A potential useful approach would be to use a single excitation wavelength to perform hyperspectral localization imaging. We report herein on the use of a variable angle tunable thin-film filter to spectrally isolate far-red emitting fluorophores. This solution was integrated into a commercial microscope platform using an open-source hardware design, enabling the rapid acquisition of SMLM images arising from fluorescence emission captured within ∼15 nm to 20 nm spectral windows (or detection bands). By characterizing intensity distributions, average intensities, and localization frequency through a range of spectral windows, we investigated several far-red emitting fluorophores and identified an optimal fluorophore pair for two-color SMLM using this method. Fluorophore crosstalk between the different spectral windows was assessed by examining the effect of varying the photon output thresholds on the localization frequency and fraction of data recovered. The utility of this approach was demonstrated by hyper-spectral super-resolution imaging of the interaction between the mitochondrial protein, TOM20, and the peroxisomal protein, PMP70.

PMID: 33379995 [PubMed - in process]



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Anti-CRISPR AcrIE2 binds the type I-E CRISPR-Cas complex but does not block DNA binding.

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Anti-CRISPR AcrIE2 binds the type I-E CRISPR-Cas complex but does not block DNA binding.

J Mol Biol. 2020 Dec 15;:166759

Authors: Mejdani M, Pawluk A, Maxwell KL, Davidson AR

Abstract
Anti-CRISPRs are protein inhibitors of CRISPR-Cas systems. They are produced by phages and other mobile genetic elements to evade CRISPR-Cas-mediated destruction. Anti-CRISPRs are remarkably diverse in sequence, structure, and functional mechanism; thus, structural and mechanistic investigations of anti-CRISPRs continue to yield exciting new insights. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AcrIE2, an anti-CRISPR that inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. Guided by the structure, we used site-directed mutagenesis to identify key residues that are required for AcrIE2 function. Using affinity purification experiments, we found that AcrIE2 binds the type I-E CRISPR-Cas complex (Cascade). In vivo transcriptional assays, in which Cascade was targeted to promoter regions, demonstrated that Cascade still binds to DNA in the presence of AcrIE2. This is the first instance of a type I anti-CRISPR that binds to a CRISPR-Cas complex but does not prevent DNA-binding. Another unusual property of AcrIE2 is that the effect of Cascade:AcrIE2 complex binding to promoter regions varied depending on the position of the binding site. Most surprisingly, Cascade:AcrIE2 binding led to transcriptional activation in some cases rather than repression, which did not occur when Cascade alone bound to the same sites. We conclude that AcrIE2 operates through a distinct mechanism compared to other type I anti-CRISPRs. While AcrIE2 does not prevent Cascade from binding DNA, it likely blocks subsequent recruitment of the Cas3 nuclease to Cascade thereby preventing DNA cleavage.

PMID: 33338493 [PubMed - as supplied by publisher]



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Subtherapeutic Photodynamic Treatment Facilitates Tumor Nanomedicine Delivery and Overcomes Desmoplasia.

Subtherapeutic Photodynamic Treatment Facilitates Tumor Nanomedicine Delivery and Overcomes Desmoplasia.

Nano Lett. 2020 Dec 10;:

Authors: Overchuk M, Harmatys KM, Sindhwani S, Rajora MA, Koebel A, Charron DM, Syed AM, Chen J, Pomper MG, Wilson BC, Chan WCW, Zheng G

Abstract
Limited tumor nanoparticle accumulation remains one of the main challenges in cancer nanomedicine. Here, we demonstrate that subtherapeutic photodynamic priming (PDP) enhances the accumulation of nanoparticles in subcutaneous murine prostate tumors ∼3-5-times without inducing cell death, vascular destruction, or tumor growth delay. We also found that PDP resulted in an ∼2-times decrease in tumor collagen content as well as a significant reduction of extracellular matrix density in the subendothelial zone. Enhanced nanoparticle accumulation combined with the reduced extravascular barriers improved therapeutic efficacy in the absence of off-target toxicity, wherein 5 mg/kg of Doxil with PDP was equally effective in delaying tumor growth as 15 mg/kg of Doxil. Overall, this study demonstrates the potential of PDP to enhance tumor nanomedicine accumulation and alleviate tumor desmoplasia without causing cell death or vascular destruction, highlighting the utility of PDP as a minimally invasive priming strategy that can improve therapeutic outcomes in desmoplastic tumors.

PMID: 33301689 [PubMed - as supplied by publisher]



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Prioritizing genes for systematic variant effect mapping.

Prioritizing genes for systematic variant effect mapping.

Bioinformatics. 2020 Dec 10;:

Authors: Kuang D, Truty R, Weile J, Johnson B, Nykamp K, Araya C, Nussbaum RL, Roth FP

Abstract
MOTIVATION: When rare missense variants are clinically interpreted as to their pathogenicity, most are classified as variant(s) of uncertain significance (VUS). Although functional assays can provide strong evidence for variant classification, such results are generally unavailable. Multiplexed assays of variant effect can generate experimental 'variant effect maps' that score nearly all possible missense variants in selected protein targets for their impact on protein function. However, these efforts have not always prioritized proteins for which variant effect maps would have the greatest impact on clinical variant interpretation.
RESULTS: Here we mined databases of clinically interpreted variants and applied three strategies, each building on the previous, to prioritize genes for systematic functional testing of missense variation. The strategies ranked genes 1) by the number of unique missense VUS that had been reported to ClinVar; 2) by movability- and reappearance-weighted impact scores, to give extra weight to reappearing, movable VUS; and 3) by difficulty-adjusted impact scores, to account for the more resource-intensive nature of generating variant effect maps for longer genes. Our results could be used to guide systematic functional testing of missense variation towards greater impact on clinical variant interpretation.
AVAILABILITY: Source code available at: https://github.com/rothlab/mave-gene-prioritization.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PMID: 33300982 [PubMed - as supplied by publisher]



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Quantifying the influence of mutation detection on tumour subclonal reconstruction.

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Quantifying the influence of mutation detection on tumour subclonal reconstruction.

Nat Commun. 2020 12 07;11(1):6247

Authors: Liu LY, Bhandari V, Salcedo A, Espiritu SMG, Morris QD, Kislinger T, Boutros PC

Abstract
Whole-genome sequencing can be used to estimate subclonal populations in tumours and this intra-tumoural heterogeneity is linked to clinical outcomes. Many algorithms have been developed for subclonal reconstruction, but their variabilities and consistencies are largely unknown. We evaluate sixteen pipelines for reconstructing the evolutionary histories of 293 localized prostate cancers from single samples, and eighteen pipelines for the reconstruction of 10 tumours with multi-region sampling. We show that predictions of subclonal architecture and timing of somatic mutations vary extensively across pipelines. Pipelines show consistent types of biases, with those incorporating SomaticSniper and Battenberg preferentially predicting homogenous cancer cell populations and those using MuTect tending to predict multiple populations of cancer cells. Subclonal reconstructions using multi-region sampling confirm that single-sample reconstructions systematically underestimate intra-tumoural heterogeneity, predicting on average fewer than half of the cancer cell populations identified by multi-region sequencing. Overall, these biases suggest caution in interpreting specific architectures and subclonal variants.

PMID: 33288765 [PubMed - indexed for MEDLINE]



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Bead-based multiplex detection of dengue biomarkers in a portable imaging device.

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Bead-based multiplex detection of dengue biomarkers in a portable imaging device.

Biomed Opt Express. 2020 Nov 01;11(11):6154-6167

Authors: Yuan X, Garg S, Haan K, Fellouse FA, Gopalsamy A, Tykvart J, Sidhu SS, Varma MM, Pal P, Hillan EM, Dou JJ, Aitchison JS

Abstract
Dengue is one of the most rapidly spreading mosquito-borne viral diseases in the world. Differential diagnosis is a crucial step for the management of the disease and its epidemiology. Point-of-care testing of blood-borne dengue biomarkers provides an advantageous approach in many health care settings, and the ability to follow more than one biomarker at once could significantly improve the management of the disease. Bead-based multiplex technologies (suspension array) can measure multiple biomarker targets simultaneously by using recognition molecules immobilized on microsphere beads. The overarching objective of our work is to develop a portable detection device for the simultaneous measurement of multiple biomarkers important in dengue diagnosis, monitoring and treatment. Here, we present a bead-based assay for the detection of one of the four serotypes of dengue virus non-structural protein (DENV-NS1) as well as its cognate human IgG. In this system, the fluorescent microspheres containing the classification fluorophore and detection fluorophore are imaged through a microfluidic chip using an infinity-corrected microscope system. Calibration curves were plotted for median fluorescence intensity against known concentrations of DENV-NS1 protein and anti-NS1 human IgG. The limit of quantitation was 7.8 ng/mL and 15.6 ng/mL, respectively. The results of this study demonstrate the feasibility of the multiplex detection of dengue biomarkers and present its analytical performance parameters. The proposed imaging device holds potential for point-of-care testing of biomarkers on a highly portable system, and it may facilitate the diagnosis and prevention of dengue as well as other infectious diseases.

PMID: 33282481 [PubMed]



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Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

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Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Mol Cell. 2020 12 17;80(6):1104-1122.e9

Authors: Hekman RM, Hume AJ, Goel RK, Abo KM, Huang J, Blum BC, Werder RB, Suder EL, Paul I, Phanse S, Youssef A, Alysandratos KD, Padhorny D, Ojha S, Mora-Martin A, Kretov D, Ash PEA, Verma M, Zhao J, Patten JJ, Villacorta-Martin C, Bolzan D, Perea-Resa C, Bullitt E, Hinds A, Tilston-Lunel A, Varelas X, Farhangmehr S, Braunschweig U, Kwan JH, McComb M, Basu A, Saeed M, Perissi V, Burks EJ, Layne MD, Connor JH, Davey R, Cheng JX, Wolozin BL, Blencowe BJ, Wuchty S, Lyons SM, Kozakov D, Cifuentes D, Blower M, Kotton DN, Wilson AA, Mühlberger E, Emili A

Abstract
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

PMID: 33259812 [PubMed - indexed for MEDLINE]



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diaPASEF: parallel accumulation-serial fragmentation combined with data-independent acquisition.

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diaPASEF: parallel accumulation-serial fragmentation combined with data-independent acquisition.

Nat Methods. 2020 Dec;17(12):1229-1236

Authors: Meier F, Brunner AD, Frank M, Ha A, Bludau I, Voytik E, Kaspar-Schoenefeld S, Lubeck M, Raether O, Bache N, Aebersold R, Collins BC, Röst HL, Mann M

Abstract
Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.

PMID: 33257825 [PubMed - in process]



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